Title

Author

Date

June 2011

Document Type

Dissertation

Degree Name

Ph.D.

Institution

Oregon Health & Science University

Abstract

For any species to survive, it must be able to respond to its environment, responses that are mediated by changes in the output of neural networks. Plasticity within neural networks, which is thought to underlie memory formation, arises from changes in the way individual neurons respond to the input they receive. The cyclic AMP response element binding protein (CREB) is a transcription factor that activates genes in response to neuronal activity and is necessary for long term potentiation (LTP). CREB activation increases the expression of several microRNAs. microRNAs are small RNA molecules that post-transcriptionally regulate gene expression. This dissertation is focused on identifying the in vivo function of one CREB-regulated microRNA locus, the miR-212/132 locus, which has been implicated in neural plasticity in vitro. Newborn neurons in the dentate gyrus of the adult hippocampus rely upon CREB signaling for their differentiation into mature granule cells and their integration into the dentate network. CREB activation increases the expression of miR-132 and miR-212. In cultured cortical and hippocampal neurons, miR-132 functions downstream from CREB to mediate activitydependent dendrite growth and spine formation in response to a variety of signaling pathways. To investigate whether miR-132 and/or miR-212 contribute to the maturation of dendrites in newborn neurons in the adult hippocampus, we inserted LoxP sites surrounding the miR-212/132 locus and specifically targeted its deletion by stereotactically injecting a retrovirus expressing Cre recombinase. Deletion of the miR-212/132 locus caused a dramatic decrease in dendrite length, arborization, and spine density. The miR-212/132 locus may express up to four distinct microRNAs, mir-132 and -212, and their reverse strands, miR-132* and -212*. Using ratiometric microRNA sensors, we determined that miR-132 is the predominantly active product in hippocampal neurons. We conclude that miR-132 is required for normal dendrite maturation in newborn neurons in the adult hippocampus and suggest that this microRNA also may participate in other examples of CREB-mediated signaling. Selective loss of miR-212/132 decreased dendrite outgrowth and spine formation in newborn neurons. However, the expression of miR-212/132 in other brain regions and the impact of miR-212/132 germline knockout (GKO) on adult neurogenesis and behavior remain unknown. We report that miR-132 is expressed in a rostral to caudal pattern in the brain of wild-type mice, and that the absolute level of miR-132 is higher than miR-212. Additionally, the loxP sites and neomycin cassette in floxed miR-212/132 mice attenuated miR-212/132 expression, but the neomycin cassette, not the reduced miRNA expression, reduced dendrite outgrowth in floxed mice. GKO mice have no deficit in dendrite outgrowth of newborn hippocampal neurons, including following seizures. Although GKO mice have normal proliferation within the dentate gyrus, the survival of newborn neurons is decreased compared to wild-type mice. Behaviorally, the GKO mice perform equivalent to wild-type mice in the open field test, the novel object test, a spatial learning task, context and cue-dependent fear conditioning, fear memory retrieval, and fear memory extinction paradigms. However, injection of HSV-GFP-Cre into the nucleus accumbens of floxed miR-212/132 mice increased cocaine-induced conditioned place preference, suggesting the miR-212/132 contributes in the rewarding properties of cocaine. Finally, I present in vitro evidence that Sprouty1 is a direct miR-132 target. Together, these studies demonstrate that miR-132 promotes dendrite outgrowth, spine formation, and survival of newborn neurons in vivo and that products of the miR-212/132 locus contribute to the rewarding properties of cocaine.

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